JP5857503B2 - Biomarkers for stress-related diseases - Google Patents

Description

  The present invention relates to therapeutic drugs for stress-related diseases such as irritable bowel syndrome, evaluation of pharmacological responsiveness and early prediction of efficacy, and the types and dosages of these therapeutic drugs that are effective for each patient. It is related with the biomarker of the stress-related disease used in order to perform selection.

  Irritable bowel syndrome (hereinafter also referred to as IBS) is a kind of gastrointestinal stress disease caused by strong tension, anxiety, stress and the like. Irritable bowel syndrome is usually a disorder of gastrointestinal motility in the large intestine, sometimes in the small intestine, such as chronic diarrhea or constipation with abdominal discomfort or abdominal pain, abdominal bloating or dull pain due to excessive gas in the intestine The main symptom is a stool abnormality such as a symptom of accumulating gas or a symptom of repeating them alternately.

  In most cases of irritable bowel syndrome, no abnormal findings of the intestine can be found in radiography or endoscopy, and there are no abnormalities in the examination items in blood tests, urinalysis, and stool tests. Abnormal symptoms persist. Such irritable bowel syndrome is a troublesome disease that has no reliable testing method and must be diagnosed only from symptoms, and even its biomarker has not been known for a long time.

  Recently, irritable bowel syndrome has been found to be a gastrointestinal disorder in which the functional relationship between the brain and intestine is central to the pathology. Non-patent document 1 describes that adenocorticotropin hormone (ACTH) is released from the pituitary gland and intestinal motility is caused by the load of corticotropin releasing hormone (CHR), which is abundant in both the brain and intestine. Corticotropin-releasing hormone and adenocorticotropin hormone have been disclosed as biomarkers.

  Furthermore, Non-Patent Document 2 discloses blood cytokines as biomarkers for irritable bowel syndrome.

  Further, in Patent Document 1, by detecting the presence or value of a diagnostic marker such as IL-8 or a cytokine such as leptin in an individual administered with a drug useful for treating irritable bowel syndrome, A method for determining a diagnostic marker profile and using an algorithm based thereon to classify whether it is associated with irritable bowel syndrome or to monitor the effect of a drug is disclosed.

  Patent Document 2 discloses functional gastrointestinal tract disorders such as irritable bowel disease by measuring neuroendocrine gastrointestinal system components that are neurohormones such as chromogranin A released from human mucosa and calculating their concentrations. A method for diagnosing gastrointestinal tract disorders is disclosed.

  Patent Document 3 discloses a method of collecting blood from an individual and measuring β-tryptase content from the serum in order to diagnose irritable bowel syndrome.

  The biomarkers described in these non-patent documents and patent documents are proteins and peptides that have a relatively high molecular weight and are difficult to be excreted in biological samples, particularly urine, and are present in blood in trace amounts and are unstable to heat. Therefore, it is difficult to detect quickly and accurately with a simple method using a simple general-purpose analytical instrument, and there are large individual differences, daily variations and reproducible variations within the same individual, and measurement errors due to high molecular weight. There is a problem that it is difficult to accurately reflect the disease state because it is largely lacking in quantitativeness.

Gut, 2004, 53, p. 1102-1108 Gastroenterology, 2006, 130, p.304-311

Special table 2010-506144 Japanese translation of PCT publication 2010-503827 International Publication No. 2010/151699

  The present invention has been made to solve the above problems, and is a non-peptidic low molecular weight compound that is stably present in a biological sample, and can accurately determine the pathology of stress diseases such as irritable bowel syndrome. The purpose is to provide a biomarker that can be reflected quickly and accurately by a simple method using a simple general-purpose analytical instrument, is difficult to decompose under detection conditions, and is used as a therapeutic agent for stress diseases And It is another object of the present invention to provide a method for distinguishing test drugs from the viewpoint of suitability of therapeutic agents for stress diseases using this biomarker.

The biomarker for stress disease for achieving the above object is detected from, for example, a sample composed of a biological fluid such as urine, blood, saliva or cerebrospinal fluid collected from a mammal suffering from stress disease [1]. A biomarker for stress-related diseases,
M / z value in negative or positive charge electroion spray mass spectrometry is
289.1, 496.7, 305.1, 291.1, 379.2, 393.2, 293.1, 760.3, 715.2, 357.1, 732.2,
407.2, 642.2, 303.1, 805.4, 305.1, 629.3, 683.3, 605.2, 307.1, 461.1, 275.1,
291.1, 263.0, 261.0, 632.4, 312.2, 255.1, 363.2, 277.1, 294.2, 331.2, 409.2,
385.2, 279.2, 343.1, 286.2, and 316.2
A biomarker for stress-related diseases, characterized in that the biomarker is at least one compound selected from the metabolic compounds and precursor compounds thereof .

で表される化合物；
下記化学式（３）

で表される化合物；
下記化学式（４）並びに（５）

で表される化合物；
３−メトキシ−４−ヒドロキシフェニルグリコール類、及び下記化学式（６）

で表される３−メトキシ−４−ヒドロキシフェニルグリコール類の硫酸抱合体；
ホモバニリン酸、及び下記化学式（７）

で表されるそれの硫酸抱合化合物；
下記化学式（８）

（式（８）中、−ＣＯ−Ｒ^１は、オクテノイル基、デカジエノイル基、イソ−デカジエノイル基、デカエノイル基及びゲラノイル基から選ばれる不飽和アシル基、又はそれらの飽和アシル基を表す。）で表されるアシル化カルニチン類；
下記化学式（９）

で表されるプロゲスタゲン類縁体；
下記化学式（１０）

（式（１０）中、Ｒ^２は水素原子、又はメチル基を表す。）で表されるベンゾキノン類；
の何れかであるというものである。
前記の目的を達成するためになされた本発明は、
〔２−２〕前記化学式（２）で表される化合物；
及び前記化学式（６）で表される３−メトキシ−４−ヒドロキシフェニルグリコール類の硫酸抱合体；の何れかであることを特徴とする過敏性腸症候群（以下、単に、ストレス性疾患と言うことがある）のバイオマーカー； More specifically, the biomarker for stress disease for achieving the above-mentioned purpose is as follows:
[ 2-1 ] Chemical formulas (1) and (2) below

In represented Ru of compounds;
The following chemical formula (3)

In represented Ru of compounds;
The following chemical formulas (4) and (5)

In represented Ru of compounds;
3-methoxy-4-hydroxyphenyl glycols and the following chemical formula (6)

A sulfated conjugate of 3-methoxy-4-hydroxyphenyl glycols represented by:
Homovanillic acid and the following chemical formula (7)

Its sulfate conjugate compound represented by:
The following chemical formula (8)

(In formula (8), —CO—R ¹ represents an unsaturated acyl group selected from an octenoyl group, a decadienoyl group, an iso-decadienoyl group, a decaenoyl group and a geranoyl group, or a saturated acyl group thereof). Acylated carnitines;
The following chemical formula (9)

A progestagen analog represented by :
The following chemical formula (10)

(In formula (10), R ² represents a hydrogen atom or a methyl group);
Either .
The present invention, which has been made to achieve the above object,
[2-2] Compound represented by the chemical formula (2);
And Formula sulfate conjugates of 3-methoxy-4-hydroxyphenyl glycols represented by (6); irritable bowel syndrome characterized in that either (hereinafter, simply be referred to as stress-induced disease biomarkers of there);

[3] The biomarker for stress disease according to [1], wherein the irritable bowel syndrome is a kind of digestive system stress disease ;

で表される化合物；
である。
ストレス性疾患のバイオマーカーは、
〔５〕下記化学式（１０）

（式（１０）中、Ｒ^２は水素原子、又はメチル基を表す。）で表される化合物；
であってもよい。 [4] Chemical formula (2)

A compound represented by:
It is.
Biomarkers for stress-related diseases
[5] Chemical formula (10)

(In formula (10), R ² represents a hydrogen atom or a methyl group);
It may be.

The present invention made to achieve the above object
Urine (6) irritable bowel syndrome is suffering from study drug was taken from a mammal which has been administered, blood, a sample consisting of a biological fluid of saliva or cerebrospinal fluid, suffering from irritable bowel syndrome for biological sample selected from a sample mammalian organs or tissues, or test drug into cells taken from any of them is added, the concentration or presence of a biomarker previously described Symbol [2-2] A method of distinguishing the test drug as a therapeutic agent for irritable bowel syndrome for the mammal by performing measurement as an index;

[7] The method according to the invention, wherein the sensitivity and insensitivity of the test drug to the mammal are selected according to the concentration of the biomarker, or the effective dose of the test drug to the mammal is estimated. [6] The method according to

[8] The method according to [6] or [7] above, wherein the index is an increase or decrease compared to the concentration of the biomarker in a sample derived from a living organism of the same healthy mammal.

[9] The method according to any one of [6] to [8] above, wherein the test drug is a mitochondrial benzodiazepine receptor antagonist;

[10] The method according to any one of [6] to [9], wherein the irritable bowel syndrome is a kind of digestive system stress disease ;

[11] The method according to any one of [6] to [10], wherein the measurement is mass spectrometry measurement, liquid chromatography measurement, gas chromatography measurement, and / or nuclear magnetic resonance spectrum measurement ;as well as

[12] Urine, blood, saliva, or cerebrospinal fluid biological fluid collected from a mammal suffering from irritable bowel syndrome , and an organ or tissue extracted from the mammal, or cells or living collected from any of them for biological sample selected from a body fluid component, as an indicator the concentration or presence of a biomarker previously described Symbol [2-2], liquid chromatography, gas chromatography, mass spectrometry, nuclear magnetic resonance spectroscopy Or a method for measuring a biomarker of irritable bowel syndrome by a measurement method combining any of them;
It is.

  The biomarker for stress disease of the present invention is a stress disease such as irritable bowel syndrome such as irritable bowel syndrome, such as recurrent low temperature stress non-human mammal which is a stress disease disease model and irritable bowel syndrome. A biometabolic compound that can accurately reflect the pathology of a diseased patient depending on the concentration or presence / absence in a sample derived from a non-human mammal or urine / blood, organ or tissue, or cell or biological fluid component thereof It is.

  This biomarker is a non-peptidic low molecular weight compound that is stably present at a sufficient concentration that can be analyzed in a sample derived from a living body, particularly in urine, and is easy to detect under analytical conditions. This biomarker can be measured quickly, accurately, qualitatively, and quantitatively by a simple method using a simple general-purpose analytical instrument, particularly a chromatograph apparatus, mass spectrometer, nuclear magnetic resonance spectrum measuring apparatus, etc. Can be detected.

  Therefore, this biomarker can be used to distinguish the test drug from the viewpoint of whether or not the test drug is suitable as a therapeutic agent for stress diseases.

  According to the method of distinguishing as a therapeutic agent for stress-related diseases for mammals of the present invention, the pharmacological responsiveness / pharmacological effect of a test drug in a non-human mammal can be evaluated qualitatively or quantitatively in vivo or in vitro. It is possible to accurately evaluate the sensitivity and insensitivity of the test drug in non-human mammals and individual patients, or to accurately estimate the effective dose of the test drug for each patient.

  Therefore, according to this distinction method, it is useful for medical treatment in which the optimal test drug is distinctly administered to a patient, or an appropriate dose is determined and administered, the therapeutic effect is estimated, and the degree of healing is judged.

  Further, according to the method for measuring a biomarker for stress-related diseases of the present invention, a specific biomarker can be accurately detected in a short time from a precious and trace amount of a biological sample, This makes it possible to supply useful data when determining the therapeutic effect of stress-related diseases and the suitability of the test drug.

  According to this method of measuring a biomarker for stress disease, the measurement sensitivity is excellent, so that the physical and mental burdens of non-human mammals and patients can be reduced by minimizing the collection of urine and blood.

It is a figure which shows an example of the identification process of the biomarker of the stress disease which applies this invention.

  Hereinafter, although the form for implementing this invention is demonstrated in detail, the scope of the present invention is not limited to these forms.

  The biomarker for stress-related diseases of the present invention is an experimental system that accurately reflects the pathology of patients with stress-related diseases such as irritable bowel syndrome. Mitochondrial benzodiazepine receptor (hereinafter also referred to as MBR) using an animal model that is repeatedly exposed to and subjected to repeated cold stress (hereinafter also referred to as RCS), or an animal model that is subjected to restraint stress using a restraint stress cage. When an antagonist is administered as a test drug, the compound specifically increases or decreases in the mammal's urine, and increases specifically in response to repeated cold stress or restraint stress. Or a decreasing compound. The biomarker is present in various biological fluids such as urine, blood, saliva, or cerebrospinal fluid of mammals, particularly humans.

で表される３−ヒドロキシ−５−メガスチグメン−９−オン類（（4-(4-hydroxy-2,6,6-trimethylcyclohex-1-en-1-yl)butan-2-one、又は3-hydroxy-5-megastigmene-9-one：以下にＨＭＯとも称する）やその硫酸抱合化合物（3,5,5-trimethyl-4-(3-oxobutyl)cyclohex-3-en-1yl hydrogen sulfate、又は3-hydroxy-5-megastigmene-9-one 3-O-sulfate：以下にＨＭＯＳとも称する）である。化学式（１）及び（２）の化合物は、抗酸化物質であるカロテノイドの一種であるβ−カロテンに由来する代謝物であり、ＲＣＳの負荷により上昇し、ＭＢＲ受容体拮抗薬投与によりそれに応答して上昇抑制する性質を有するものである。化学式（１）及び（２）の化合物は、光学活性体であってもよいがｄｌ体であってもよい。 Preferred examples of such biomarkers for stress diseases include the following chemical formulas (1) and (2)

3-hydroxy-5-megagustigment-9-one ((4- (4-hydroxy-2,6,6-trimethylcyclohex-1-en-1-yl) butan-2-one or 3- hydroxy-5-megastigmene-9-one: hereinafter also referred to as HMO) and its sulfate-conjugated compound (3,5,5-trimethyl-4- (3-oxobutyl) cyclohex-3-en-1yl hydrogen sulfate, or 3- Hydroxy-5-megastigmene-9-one 3-O-sulfate (hereinafter also referred to as HMOS) The compounds of the chemical formulas (1) and (2) are added to β-carotene, which is a kind of antioxidant carotenoid. It is a metabolite derived from the compound, and has the property of being increased by the load of RCS and suppressing the increase in response to administration of the MBR receptor antagonist, The compounds of the chemical formulas (1) and (2) are optically active substances. However, it may be a dl form.

  Similar to the compounds of the chemical formulas (1) and (2), as a biomarker for stress-related diseases having this property, a reduced compound in which the double bond of the unsaturated group and / or the carbonyl group is reduced, for example, the unsaturated compound A compound in which a group is reduced to a single bond and may be a diastereomer or a mixture thereof, or a compound in which a carbonyl group is reduced to a hydroxy group and may be a diastereomer or a mixture thereof. A hydroxyl group-substituted compound in which a hydroxyl group is introduced at any of the 2, 4, 6, 7, or 8 positions of these reducing compounds may be used.

で表されカロテノイド類に由来する代謝物である３−ヒドロキシ−５−メガスチグメン−９−オン類のグルクロン酸抱合化合物（3,5,5-trimethyl-4-(3-oxobutyl)cyclohex-3-en-1-yl hexopyranosiduronic acid、又は3-O-Glucronide 3-hydroxy-5-megastigmene-9-one）であってもよい。そのアグリコンは光学活性体であってもよいがｄｌ体であってもよい。 In addition, biomarkers for stress diseases are represented by the following chemical formula (3)

A glucuronide-conjugated compound of 3-hydroxy-5-megastigmen-9-ones, which is a metabolite derived from carotenoids (3,5,5-trimethyl-4- (3-oxobutyl) cyclohex-3-en -1-yl hexopyranosiduronic acid or 3-O-Glucronide 3-hydroxy-5-megastigmene-9-one). The aglycone may be an optically active form or a dl form.

で表される２−カルボキシエチル−６−ヒドロキシクロマン類（3-[(2S)-2,5,7,8-tetramethyl-6-(hydroxy)-3,4-dihydro-2H-chromen-2-yl]propanoic acid：以下にα−ＣＥＨＣとも称する）、及びそれの硫酸抱合化合物（3-[(2S)-2,5,7,8-tetramethyl-6-(sulfooxy)-3,4-dihydro-2H-chromen-2-yl]propanoic acid：以下にα−ＣＥＨＣＳとも称する）であってもよい。α−ＣＥＨＣやその硫酸抱合化合物α−ＣＥＨＣＳの位置異性体やその類縁体、例えばβ−ＣＥＨＣやβ−ＣＥＨＣＳ、γ−ＣＥＨＣやγ−ＣＥＨＣＳ、δ−ＣＥＨＣやδ−ＣＥＨＣＳ、又はそれらの何れかの混合物であってもよい。その硫酸抱合化合物α−、β−、γ−又はδ−ＣＥＨＣＳの二量体、若しくはそれ以外の分子イオンとの複合体となったものであってもよい。また、同じくビタミンＥ由来のもので、ＣＥＨＣやＣＥＨＣＳの前駆化合物である3,4-dihydro-6-hydroxy-α,2,5,7,8-pentamethyl-2H-1-benzopyran-2-pentanoic acid （α−ＣＭＢＨＣ）、その異性体であるβ−、γ−又はδ−ＣＭＢＨＣや、それらの硫酸抱合化合物であるβ−、γ−又はδ−ＣＭＢＨＣＳであってもよい。 The biomarker for stress disease is derived from vitamin E and has the following chemical formulas (4) and (5)

2-carboxyethyl-6-hydroxychromans (3-[(2S) -2,5,7,8-tetramethyl-6- (hydroxy) -3,4-dihydro-2H-chromen-2- yl] propanoic acid: hereinafter also referred to as α-CEHC) and its sulfate-conjugated compound (3-[(2S) -2,5,7,8-tetramethyl-6- (sulfooxy) -3,4-dihydro- 2H-chromen-2-yl] propanoic acid: hereinafter also referred to as α-CEHCS). α-CEHC and its sulfate conjugated compound α-CEHCS positional isomers and analogs thereof, such as β-CEHC and β-CEHCS, γ-CEHC and γ-CEHCS, δ-CEHC and δ-CEHCS, or any of them It may be a mixture of It may be a dimer of the sulfate-conjugated compound α-, β-, γ-, or δ-CEHCS, or a complex with other molecular ions. Also, 3,4-dihydro-6-hydroxy-α, 2,5,7,8-pentamethyl-2H-1-benzopyran-2-pentanoic acid, which is also derived from vitamin E and is a precursor compound of CEHC and CEHCS (Α-CMBHC), its isomers β-, γ-, or δ-CMBHC, and their sulfate-conjugated compounds β-, γ-, or δ-CMBHCS may be used.

で表される３−メトキシ−４−ヒドロキシフェニルグリコールの硫酸抱合体（2-hydroxy-1-(4-hydroxy-3-methoxyphenyl)ethyl hydrogen sulfate：以下にＭＨＰＧＳとも称する）であってもよい。 Biomarkers for stress diseases are derived from catecholamines, such as 3-methoxy-4-hydroxyphenylglycol (hereinafter also referred to as MHPG) and its sulfate conjugates, for example, the following chemical formula ( 6)

Or 2-methoxy-1-hydroxyphenyl glycol sulfate (2-hydroxy-1- (4-hydroxy-3-methoxyphenyl) ethyl hydrogen sulfate: hereinafter also referred to as MHPGS).

で表されるそれの硫酸抱合化合物（[3-methoxy-4-(sulfooxy)phenyl]acetic acid：以下にＨＶＡＳとも称する）であってもよい。 The biomarker for stress disease may be homovanillic acid derived from catecholamine (hereinafter also referred to as HVA), which has the following chemical formula (7)

It may be a sulfate-conjugated compound ([3-methoxy-4- (sulfooxy) phenyl] acetic acid: hereinafter also referred to as HVAS).

例えば、−ＣＯ−Ｒ^１が不飽和アシル基として、オクテノイル基であるカルニチン類（Octenolycarnitine：以下にC8:1-Carnitineとも称する）、デカジエノイル基やイソデカジエノイル基やゲラノイル基であるカルニチン類（2-trans,4-cis-Decadienoylcarnitineなど：以下にC10:2-Carnitineとも称する）であってもよく、−ＣＯ−Ｒ^１が飽和アシル基としてそれらに対応する飽和アシル基、例えばデカノイル基であるカルニチン類（Decanoylcarnitineなど：以下にC10:0-Carnitineとも称する）であってもよい。
ストレス性疾患のバイオマーカーは、コレステロールに由来するプロゲスタゲン類縁体であって、下記化学式（９）

で表される4-Pregnen-19-ol-3,20-dione（以下にC21 Progestageneとも称する）であってもよく、後述する表５に記載しているようなそれの水酸基の位置が異なる位置異性体やその構造異性体である各種Pregnen類やAndrostadien類やAndrosten類やEstren類であってもよい。 The biomarker for stress disease is derived from carnitine and has the following chemical formula (8)

For example, a -CO-R ¹ is an unsaturated acyl group, carnitines are octenoyl group (Octenolycarnitine: below C8: 1-Carnitine also called), Dekajienoiru group and isobutyl deca diene decanoyl group and carnitines is Geranoiru group ( 2-trans, 4-cis-Decadienoylcarnitine and the like: hereinafter also referred to as C10: 2-Carnitine), and —CO—R ¹ is a saturated acyl group corresponding to them as a saturated acyl group, for example, a decanoyl group Carnitines (such as Decanoylcarnitine: hereinafter also referred to as C10: 0-Carnitine) may be used.
A biomarker for stress-related diseases is a progestagen analog derived from cholesterol having the following chemical formula (9)

4-Pregnen-19-ol-3,20-dione (hereinafter also referred to as C21 Progestagene) represented by the above, and the position of the hydroxyl group as described in Table 5 to be described later is different. It may be an isomer or various structural isomers such as Pregnen, Androstadien, Androsten, and Estren.

（式（１０）中、Ｒ^２は水素原子、又はメチル基を表す。）で表されるベンゾキノン類であってもよい。化学式（１０）の化合物は、光学活性体であってもよいが、ｄｌ体であってもよく、ジアステレオマー又はその混合物であってもよい。 The biomarker for stress-related diseases has the following chemical formula (10)

(In formula (10), R ² represents a hydrogen atom or a methyl group.) A benzoquinone represented by the formula (10) may be used. The compound of the chemical formula (10) may be an optically active form, but may be a dl form, a diastereomer or a mixture thereof.

Biomarkers of stress disorders, and the said m / z, it is a compound identified by the example C ₁₈ modified silica gel-based filler or a polymer-based liquid chromatography retention time by fillers R _t, may. Here, the liquid column chromatography can be performed under UPLC conditions described later, and the measurement conditions may be appropriately changed. Most of these biomarkers are increased by RCS and have the property of suppressing the increase in response to MBR antagonist administration. On the contrary, m / z is 632.4 and retention time is 85 seconds. Only these compounds have the property of being decreased by the load of RCS and suppressing the decrease in response to MBR antagonist administration.

Test drugs have shown examples of mitochondrial benzodiazepine receptor (also known as translocator protein 18 KDa (TSPO)) antagonists for irritable bowel syndrome, but drugs for other stress diseases such as anti-anxiety drugs (for example, Benzodiazepine anxiolytics, thienodiazepine anxiolytics, non-benzodiazepine anxiolytics, serotonin agonists, CRF antagonists, etc., antidepressants (eg monoamine liberators, monoamine oxidase inhibitors, monoamine reuptake inhibitors ( SNRI, SSRI), CRF antagonist, neurotensin antagonist, tricyclic antidepressant, tetracyclic antidepressant, etc.), anticholinergic agent, gastrointestinal function regulator (eg, intestinal regulating agent, CCK-A antagonist) Drugs, neurotensin antagonists, opioid agonists, muscarinic agonists, 5-HT ₄ agonists Etc.), gastrointestinal motility promoting agents (eg, intestinal regulating agents, CCK-A antagonists, neurotensin antagonists, opioid agonists, muscarinic agonists, 5-HT ₄ agonists, etc.), antidiarrheal agents (eg, diarrhea) Drugs, opioid μ receptor stimulants, etc.), laxatives (eg, expansive laxatives, salt laxatives, stimulant laxatives, affinity polyacrylic resins, rubiprostone, etc.), mucosal paralytics, autonomic nerve regulators, calcium antagonists, It may be a phosphodiesterase inhibitor, serotonin antagonist (for example, 5-HT ₃ antagonist, 5-HT ₄ antagonist), darifadazine, polycarbophil calcium, lactic acid bacteria preparation, antibiotic (for example, rifaximin).

  Biomarkers of this stress disorder are digestive system, central nervous system, respiratory system, circulatory system, urinary system, genital system, gynecological system, endocrine system, metabolic system, ophthalmic system, otolaryngology system caused by stress It can be an indicator component specific to various diseases such as oral system, dental system, skin system, orthopedics / surgical system. It is particularly useful as an indicator component for patients with gastrointestinal stress diseases such as irritable bowel syndrome.

  Although this biomarker for stress-related diseases has been shown to be detected from mammalian urine as a biological sample, it is collected from blood, organs and / or tissues of mammals, particularly stress-related diseases, as necessary. Detection is also possible from a sample derived from a living body such as a cultured cell. From the viewpoint of reducing the physical and mental burdens of non-human mammals and patients by repeated urine collection and blood collection, and securing a sufficient amount or storing the sample, it is preferable to use urine as a biological sample.

  The biomarker for stress disease is preferably one or more selected from among these, and more preferably about two or more types in order to improve detection accuracy and reliability. As the plurality of biomarkers, any combination of the compound of formula (2), the compound of formula (3), homovanillic acid, and a sulfated compound of 3-methoxy-4-hydroxyphenylglycol is preferable.

Biomarkers of these stress-related diseases include urine, blood, saliva or cerebrospinal fluid, organs or tissues, or presence / identification and concentration in biological samples collected from those cells, various instrumental analysis, specifically, Physicochemical physicochemical analysis, more specifically,
Chromatographic methods such as high performance liquid chromatography (HPLC), ultra high performance liquid chromatography (UHPLC), supercritical liquid chromatography (SFC), gas chromatography (GC);
Mass spectrometry (MS method), for example, electron ionization method (EI-MS method), chemical ionization method (CI-MS method), electrolytic desorption method (FD-MS method), fast atom collision method (FAB-MS method) Magnetic field deflection type, quadrupole type (QMS) by matrix assisted laser desorption ionization method (MALDI-MS method), electrospray ionization method (ESI-MS method), atmospheric pressure chemical ionization method (APCI-MS method), etc. ), Triple quadrupole (TQ-MS), ion trap (IT-MS), time of flight (TOF-MS), Fourier transform ion cyclotron resonance (FI-ICR-MS), tandem (MS) / MS) method;
Nuclear magnetic resonance spectrum measurement method (NMR method), for example, ¹ H-NMR or ¹³ C-NMR, more specifically, one-dimensional ¹ H-NMR or ¹³ C-NMR, HH- or CH-COSY, NOESY, TOCSY, two-dimensional NMR such as wetTOCSY;
Or any combination thereof, for example, liquid chromatography-mass spectrometry (LC-MS), preferably ultrahigh performance liquid chromatography-mass spectrometry (UPLC / MS method; UPLC is a registered trademark), high performance liquid chromatography Graph-mass spectrometry (HPLC-MS), liquid chromatograph-tandem mass spectrometry (LC-MS / MS), liquid chromatograph-nuclear magnetic resonance spectroscopy (LC-NMR method), liquid chromatograph-nucleus Magnetic resonance spectrum / mass spectrometry (LC-NMR / MS)
Therefore, it is detected qualitatively or quantitatively as compared with a normal healthy mammal.

  Other methods for detecting biomarkers of stress diseases include biochemical physicochemical analysis, specifically, EIA method (Enzyme Immunoassay) using antigen-antibody reaction, especially ELISA method (Enzyme-linked). Immunosorbent Assay (enzyme-linked immunosolvent assay), RIA (Radio-immuno-assay) and the like can also be used.

  Biomarkers for stress-related diseases in biological samples can be identified by comparing the mass spectrometry spectrum and nuclear magnetic resonance spectrum of the sample, and comparing the retention times of the chromatograms of the sample. The concentration of these can be calculated by comparing the intensity of these spectra or the peak area of the chromatogram with the standard calibration curve of the intensity or peak area and concentration in the standard.

  Such biomarkers of stress diseases are analyzed by the following metabolome analysis that selects specific substances as markers by analyzing data obtained by comprehensive analysis from a large number of components contained in biological samples, It was narrowed down and identified as a useful marker component. The identification process will be described in detail with reference to FIG.

(I) First, normal non-human mammals not suffering from stress-related diseases were preliminarily raised.

(Ii) The first urine collection is performed from this mammal, urinary metabolites are extracted from the urine, and all the components are subjected to ultra high performance liquid chromatography-mass spectrometry (UPLC / TOF-MS method), and in positive mode. M / z is measured in negative mode, and matrix data is created based on the raw data of the retention time, m / z, and relative intensity of each of the thousands components obtained. From the data, a plot graph is created with the horizontal axis as the retention time and the vertical axis as m / z.

(Iii) In addition, by preliminarily rearing a mammal of the same kind as that of the same species in the daytime by alternately exposing to a low temperature and a room temperature every hour for several days, the RCS load is intentionally applied to this mammal. Call. Since this stress load is very close to the pathological condition of human stress disease, particularly irritable bowel syndrome, it can be an animal model of irritable bowel syndrome.

(Iv) After RCS loading, do not administer anything, or administer the vehicle or test substance, and then perform a second urine collection. Similarly, all the components are subjected to ultra high performance liquid chromatography-mass spectrometry, and matrix data And plot graph. The matrix data of the first urine collection and the second urine collection are compared with the plot graph, and thousands of components are primarily selected as an increase / decrease component specific to the RCS load.

(V) On the other hand, a medium or test substance is administered to a mammal not loaded with RCS or to the mammal while being loaded with RCS, and after a while, the urine is collected for the third time. Graph-mass spectrometry is applied to create matrix data and a plot graph. False positive markers such as components whose intensity increases / decreases regardless of RCS load, components whose intensity hardly increases / decreases even when the test substance is administered, and components whose intensity increase / decrease is not reproducible Exclude ingredients and secondary sort only hundreds of ingredients that increase in intensity with RCS loading and decrease in intensity with test substance administration or decrease in intensity with RCS loading and increase in intensity with test substance administration .

(Vi) Furthermore, in order to ensure quantitative measurement, remove low-intensity components (signal values) that are insensitive or affected by contaminating components, and re-administer the test substance to ensure reproducibility. The high component is subjected to quantitative analysis by using UPLC / MRM method (MS / MS analysis (MRM) using UPLC), and then the tens of components are selected as biomarkers for stress diseases. To do.

(Vii) The biomarker of this stress disease is identified by various instrumental analysis, for example, mass spectrometry, nuclear magnetic resonance spectrum measurement method and the like.

  In addition, primary to tertiary sorting is performed by multivariate analysis such as principal component analysis, regression analysis, cluster analysis, variance analysis, Welch's t-test, paired t-test, difference comparison test, or unpaired t-test as appropriate. Is called.

  The biomarker for stress disease identified as described above has a positive correlation or a negative correlation between the concentration in the biological sample and the therapeutic effect of the stress disease. For example, a correlation tendency is observed between the amount of defecation during a stress load period on a stress model animal and the biomarker concentration in urine. Therefore, based on the concentration of this biomarker, it can be converted into a numerical value by converting as a disease state level of stress-related diseases. Also, based on the concentration, the administered test drug can be converted into a numerical value by converting it as the effectiveness level of the therapeutic agent for stress disease. Moreover, it may be converted into a numerical value as the presence or absence or sensitivity level of a test drug administered to a patient with a stress disorder, and used to determine the suitability of administration of the test drug as necessary. Moreover, you may use for estimating the effective dose of a test drug with respect to a mammal, especially the patient of a stress-related disease, the optimal dose, the administration possible range, etc.

  Hereinafter, an example in which the biomarker for stress disease of the present invention is specifically identified will be described in detail.

Example 1
First, an irritable bowel syndrome animal model that is very close to the pathological condition of irritable bowel syndrome, which is said to be a stress disorder and affects several percent of the total population, is obtained by RCS loading on rats. And biomarkers for stress-related diseases were identified.

(1. Animals used and their preliminary breeding)
Male Wistar rats (Nippon Charles River Co., Ltd.) were preliminarily raised at room temperature, quarantined as necessary, and acclimatized so that they would be 7 weeks old at the start of the production of an irritable bowel syndrome animal model.

(2. Procedure for preparing animal model of irritable bowel syndrome and administration of test substance or control substance)
The specific procedure for producing an animal model of irritable bowel syndrome in which rats were loaded with RCS was as follows. On the first day, the breeding environment temperature was set to 8 ° C. and 2 ° C. between 9 am and 7 pm Change to room temperature (24 ± 2 ° C) alternately, and then raise at 2 ° C in the rearing environment temperature until the next morning. From the second day to the sixth day, the rearing environment is between 9:00 am and 8:00 pm The temperature is changed nine times every hour, and the animals are raised at a breeding environment temperature of 2 ° C. from the end of the ninth time until the next morning. This induces pathological conditions similar to those of human irritable bowel syndrome, such as increased sensitivity to rectal extension stimulation, and has become an animal model for stress-related diseases.

  Each of the two groups of rats was administered a medium which is an MBR antagonist as a test substance or a 0.5 w / v% methylcellulose aqueous solution as a control substance while applying an RCS load as described above. A vehicle administration control group was used. Test substance and vehicle dosing procedures were in the morning (between 9am and 11am) and afternoon (between 5pm and 6:30 pm) from day 1 to day 6 of RCS loading, respectively. The test substance (10 mg / kg) or vehicle (5 mL / kg) is orally administered once. In addition, as an MBR selective ligand which is a test substance, (1- (2-chlorophenyl) described in European Journal of Pharmacology, 1985, Vol. 119, p.153-167. -N-methyl-N- (1-methylpropyl) -3-isoquinolinecarboxamide) PK11195, any of the compounds of the examples described in WO 2004/113300 and WO 2006/068164 It was.

(3. Urine collection and analysis pretreatment)
a) On the day before RCS loading and on the 7th day of RCS loading, after completion of loading, the rats were placed in a metabolic cage, and after 7 hours, urine was collected from each of the rats in the non-administration group, vehicle administration group and test substance administration group All the amounts were collected and used as a non-administration-RCS load group sample, a vehicle administration-RCS load group sample, and a test substance administration-RCS load group sample, respectively.

  b) On the day before the RCS loading and the 7th day of the RCS loading, after completion of the loading, the rats are placed in a metabolic cage, and after 7 hours, the whole amount of urine is collected from each of the rats in the vehicle administration group and the test substance administration group, Respectively, a medium administration-RCS load group sample and a test substance administration-RCS load group sample before and after RCS loading were used. Vehicle administration was performed for 6 days, and a vehicle administration-non-RCS loading group was provided as a group not loaded with RCS. Also for these, samples of each group were collected as described above.

  After measuring the volume of the collected urine for each sample, the sample was centrifuged at 12,000 G for 5 minutes at room temperature, and 1/60 volume of 1.2% sodium azide aqueous solution (final concentration 0. 02%) after addition, it was frozen in liquid nitrogen and stored at −80 ° C. until analysis.

(4. Mass spectrometry)
In order to use each sample obtained in a) for analysis by the UPLC / TOF-MS method, the frozen sample was redissolved and used, and 50 μL of the collected urine contained 0.1 vol% formic acid. 150 μL of an aqueous solution was added and mixed, and the supernatant obtained by centrifugation at 16000 G for 3 minutes at 4 ° C. was filtered through a 2 μm polytetrafluoroethylene (PTFE) filter.

  It was subjected to mass spectrometry in negative mode and positive mode by UPLC / TOF-MS method system equipment. The instrument is an ACQUITY Ultra Performance Liquid Chromatography system (manufactured by Waters), and a mass spectrometer is Micromass LCT Premier (manufactured by Waters) of orthogonal acceleration time-of-flight. It is a combined system.

[Negative mode measurement conditions]
<UPLC conditions>
-Column: ACQUITY UPLC BEH C18 (1.7 μm particle size 2.1 mm inner diameter × 150 mm length) (Waters)
-Column oven temperature: 40 ° C
-Mobile phase component A: 0.1 wt% formic acid aqueous solution-Mobile phase component B: 0.1 wt% formic acid acetonitrile solution-Gradient conditions for elution of mobile phase component A / B 0 min 100% (A) 0% (B )
7 minutes 0% (A) 100% (B)
14 minutes 0% (A) 100% (B)
14.1 minutes 100% (A) 0% (B)
21 minutes 100% (A) 0% (B)
・ Flow rate: 300 μL / min ・ Autosampler temperature: 4 ° C.
・ Measurement sample injection volume: 4 μL
<MS conditions>
・ Polarity: Negative ・ Capillary voltage: 2700V
・ Cone voltage: 35V
-Spray gas temperature: 350 ° C
・ Ion source temperature: 120 ℃
・ Cone gas flow rate: 50 L / h
-Spray gas flow rate: 500 L / h
・ Mode: W mode

[Positive mode measurement conditions]
<UPLC conditions>
-Column: ACQUITY UPLC BEH C18 (1.7 μm particle size 2.1 mm inner diameter × 150 mm length) (Waters)
-Column oven temperature: 40 ° C
-Mobile phase component A: 0.1 wt% formic acid aqueous solution-Mobile phase component B: 0.1 wt% formic acid acetonitrile solution-Gradient conditions for elution of mobile phase component A / B 0 min 100% (A) 0% (B )
7 minutes 0% (A) 100% (B)
15 minutes 0% (A) 100% (B)
15.1 min 100% (A) 0% (B)
20 minutes 100% (A) 0% (B)
・ Flow rate: 300 μL / min ・ Autosampler temperature: 4 ° C.
・ Measurement sample injection volume: 4 μL
<MS measurement conditions>
・ Polarity: Positive capillary voltage: 2800V
・ Cone voltage: 44V
-Spray gas temperature: 350 ° C
・ Ion source temperature: 120 ℃
・ Cone gas flow rate: 50 L / h
-Spray gas flow rate: 600 L / h
・ Mode: W mode

  The biomarker candidate compounds narrowed down by the UPLC / TOF-MS method are further subjected to quantitative analysis by the UPLC / MRM method, and the urine collected in b) is centrifuged at 10,000 G for 5 minutes at 4 ° C. Then, 200 μL of 0.1 wt% aqueous formic acid solution was added to 50 μL of the supernatant obtained and stirred.

  API 4000QTRAP LC /, which is a triple quadrupole type with MRM (Multiple reaction Monitoring) mode, using the ACQUITY Ultra Performance Liquid Chromatography system (manufactured by Waters) as an ultra-high performance liquid chromatograph. Analysis was performed in combination with MS / MS System (Applied Biosystems).

<UPLC measurement conditions>
-Column: ACQUITY UPLC BEH C18 (1.7 μm particle size 2.1 mm inner diameter × 150 mm length) (Waters)
-Column oven temperature: 40 ° C
-Mobile phase component A: 0.1 wt% formic acid aqueous solution-Mobile phase component B: 0.1 wt% acetonitrile solution-Gradient conditions for elution of mobile phase component A / B 0 min 100% (A) 0% (B)
7 minutes 0% (A) 100% (B)
14 minutes 0% (A) 100% (B)
14.1 minutes 100% (A) 0% (B)
21 minutes 100% (A) 0% (B)
・ Flow rate: 300 μL / min ・ Measurement sample injection volume: 4 μL
<MS conditions>
・ Scan type: MRM
・ Polarity: Negative ・ Ionization method: ESI (electrospray ion) method ・ Source temperature: 400 ° C.

  Under these conditions, m / z of monitor ions at Q1 and Q3 of biomarker candidate compounds and analysis conditions are as shown in Table 1-1 and Table 1-2 below (DP: Declustering Potential, CE: Collision) Energy, CXP: Collision Cell Exit Potential).

<MS conditions>
・ Scan type: MRM
・ Polarity: Positive ・ Ionization method: ESI (electrospray ion) method ・ Source temperature: 400 ° C.

  The monitor ion m / z at Q1 and Q3 under the above conditions and the analysis conditions are as shown in Table 2 below (DP: Declustering Potential, CE: Collision Energy, CXP: Collision Cell Exit Potential) ).

  The related compounds of the candidate biomarker compounds narrowed down by UPLC / TOF-MS method are also used for quantitative analysis by UPLC / MRM method. 200 μL of a 0.1 wt% aqueous formic acid solution was added to 50 μL of the supernatant obtained by centrifugation at 0 ° C. and stirred.

  API 4000QTRAP LC /, which is a triple quadrupole type with MRM (Multiple reaction Monitoring) mode, using the ACQUITY Ultra Performance Liquid Chromatography system (manufactured by Waters) as an ultra-high performance liquid chromatograph. Analysis was performed in combination with MS / MS System (Applied Biosystems).

<UPLC measurement conditions>
Column: ACQUITY UPLC BEH C18 (1.7 μm particle size 2.1 mm inner diameter x 100 mm length) (Waters)
-Column oven temperature: 40 ° C
-Mobile phase component A: [Positive] 0.1 wt% formic acid aqueous solution, [Negative] 6.5 mM ammonium hydrogen carbonate aqueous solution-Mobile phase component B: [Positive] 0.1 wt% methanol solution, [Negative] 6.5 mM hydrogencarbonate Gradient conditions for elution of 95% aqueous ammonium solution / mobile phase component A / B 0 min 100% (A) 0% (B)
4 minutes 30% (A) 70% (B)
4.5 minutes 2% (A) 98% (B)
10 minutes 2% (A) 98% (B)
10.1 minutes 100% (A) 0% (B)
17 minutes 100% (A) 0% (B)
・ Flow rate: 350 μL / min ・ Measurement sample injection volume: 4 μL
<MS conditions>
・ Scan type: MRM
・ Polarity: Negative ・ Ionization method: ESI (electrospray ion) method ・ Source temperature: 400 ° C.

  The monitor ion m / z of Q1 and Q3 of the biomarker candidate compound under these conditions and the analysis conditions are as shown in Table 3 below (DP: Declustering Potential, CE: Collision Energy, CXP: Collision Cell Exit) Potential).

<MS conditions>
・ Scan type: MRM
・ Polarity: Positive ・ Ionization method: ESI (electrospray ion) method ・ Source temperature: 400 ° C.

  The monitor ion m / z at Q1 and Q3 of the biomarker candidate compound under these conditions and the analysis conditions are as shown in Table 4 below (DP: Declustering Potential, CE: Collision Energy, CXP: Collision Cell Exit Potential) ).

(5. Metabolome analysis)
The chromatogram data obtained by UPLC / TOF-MS analysis was converted into data in netCDF format using mass spectrometry software MassLynx Ver.4.1 (manufactured by Waters). Subsequently, the obtained data was obtained by using the analysis software MZmine Ver.1.91 (free software: obtained from http://mzmine.sourceforge.net/download.shtml). ), Elution time (RT), peak area value, and peak intensity value data were converted into a matrix format.

Using this matrix format data,
The variation ratio and p-value were calculated by using the array analysis software Expressionist Pro Analyst Ver. 5.1.4 (Genedata) or Office Excel 2007 (Microsoft).

  First, (1) (i) Welch's t-test was performed using the sample on the previous day and the sample on the 7th day of RCS loading in the non-administration-RCS loading group and the vehicle administration-RCS loading group, respectively. .2, a signal value variation ratio of 1.5 times or more or 0.67 times or less between both groups, and (ii) vehicle administration-RCS loading group and test substance administration-RCS loading group, RCS, respectively. Using the 7th day sample after loading, Welch's t-test was performed, and the peak that showed a p-value of less than 0.3 and a fluctuation ratio of 1.33 times or more or 0.75 times or less between both groups, 315 peaks were selected as biomarker candidates.

  Next, (2) the difference between the 7th day of RCS loading and the day before RCS loading (the signal value on the 7th day−the signal value on the 0th day) of the non-administration-RCS load group and the vehicle administration-RCS load group, respectively. In comparison, using Welch's t-test, p-value is less than 0.2, the signal value variation ratio is 1.5 times or more or 0.67 times or less between both groups, and vehicle administration-RCS load group The value obtained by subtracting the median value of the difference between the RCS loading day 7 and the RCS loading day of the test substance administration-RCS loading group from the median value of the difference between the RCS loading day 7 and the RCS loading day is greater than 0 379 peaks were selected as biomarker candidates.

  Since the statistical method is different between the peak selected by the procedure (1) and the peak selected by the procedure (2), it goes without saying that the peaks extracted by both methods are not limited. The peaks extracted by this method are also considered as biomarker candidates, and a total of 515 peaks were selected.

  Of the biomarker candidates selected above, UPLC-MRM quantitative measurement was separately performed on 89 peaks (negative: 62 peaks, positive: 27 peaks). For measurement data processing, Analyst Ver. 1.4.2 (Applied Biosystems) and Excel 2002 (Microsoft) were used. The Analyte Peak Area (counts) value of all peaks was corrected with the Analyte Peak Area (counts) value of creatinine as a control. Comparison of variation difference between before and after RCS loading, RCS loading or non-loading of each group with respect to samples of each group of vehicle administration-non-RCS loading group, vehicle administration-RCS loading group, and test substance administration-RCS loading group The comparison and variation ratio after 7 days were evaluated. Comparative analysis was performed by unpaired t-test (two-sided).

As a result, biomarkers that were enhanced by RCS loading and decreased by test substance administration, and biomarkers that were decreased by RCS loading and increased by test substance administration could also be selected (Table 5).
In addition, as for compounds related to the identified biomarkers, a sample was purchased and evaluated whether it could be a biomarker for stress-related diseases as described above. Α-CEHC, β and γ-CEHCS, C8: 1 It was found that carnitine (octenoylcarnitine) and C10: 0 carnitine (decanoylcarnitine) can be biomarkers (Table 6).

  As a result, a total of 38 types of compounds were found as biomarkers for stress-related diseases, as shown in Tables 5 and 6 below.

  As the presumed structure of Compound No. 2, 3-O-sulfated 3-Hydroxy-5,6-epoxy-5-megastigmene-9-dione, 3 or 6-O-sulfated 3,6-Dihydroxy-4-megastigmen- 9-one, 3 or 7-O-sulfated 3,7-Dihydroxy-5-megastigmen-9-one, 3 or 11-O-sulfated 3,11-Dihydroxy-5-megastigmen-9-one, 3 or 6- O-sulfated 3,6-Dihydroxy-7-megastigmen-9-one, or 3,6-Epoxy-7-megastigmene-5,9-diol, 3- (1-Hydroxyoctyl) -5-methyl-2 (5H) -furanone, 3-Oxo-2-pentylcyclopentaneacetic acid Me ester, 5,6-Epoxy-7-megastigmene-3,9-diol, 5,8-Epoxy-6-megastigmene-3,9-diol, 5- (3 -Ethyl-4-methyl-1-pentenyl) -4,5-dihydro-3-hydroxy-5-methyl-2 (3H) furanone, 5- (7-Hydroxy-6-methyloctyl) -2 (5H) -furanone , 6,9-Dihydroxy-4,7-megastigmadien-3-one, 7,8-Dihydro, 6,9-Dihydroxy-7-megastigmen-3-one, 6,9-Epoxy-4-megastigmene-3,9 -diol, Durgamone, Stegobiol, or their sulfate conjugates.

  As the presumed structure of Compound No. 3, 1- (3,4-Dihydroxyphenyl) -2-propen-1-ol, 3'-Me ether, 4 '-(2-methylpropanoyl), 1-Ac, 1-O -Coumaroylglycerol, 4'-Me ether, 2,3-O-isopropylidene, 11-Hydroxy-12-methoxydihydrokawain, Me ether, 11-Hydroxy-3,8-dioxo-1,4-eudesmadien-12-oic acid, Me ester, 15-Hydroxy-1 (10), 4,11 (13) -germacratrien-12,6-olid-14-oic acid, Me ester, 3- (3,4-Dihydroxyphenyl) -2-propen-1- ol, 3'-Me ether, 4'-O- (2-methylpropanoyl), 1-Ac, 3- (4-Hydroxy-3-methoxyphenyl) -2-propenoic acid, 4-Methyl-3-oxopentyl ester, 3 -(4-Hydroxy-3-methoxyphenyl) -2-propenoic acid, 4-O- (3-Oxohexyl), 5,7-Dihydroxy-2,2-dimethyl-2H-1-benzopyran-6-propanoic acid; Di -Me ether, 6,7-Epoxy-1-hydroxy-13-nor-9-eremophilene-8,11-dione, Ac, 8,9-Dihydroxy-14-oxo-1 (10), 4,11 (13 ) -germacratrien-12,6-olide, 9-Me ether, 8- (2,3-Dihydroxy-3-methylbutyl) -7-hydroxy-2H-1-benzopyran-2-one, 3 ', 7-Di- Me ether, 8-Acetyl-5,6,7-trihydroxy-2,2-dimethyl-2H-1-benzopyran; Tri-Me ether, 8-Hydroxy-1 (10), 4,7 (11) -germacratrien -12,8-olid-15-oic acid, Me ester, 8-Hydroxy-1,3,7 (11) -elematrien-12,8-olid-15-oic acid; 8-OH-form, Me ester, 8-Hydroxy-7 (11) -eremophilene-12,8: 15,6-diolide, Me ether, Cladosporin, Coriandrone A, Coriandrone B, Curvularin, Evodione, Isosecotanapartholide; 3-Epimer, Me ether, Isosecotanapartholide; Me ether, Morinin A; 3-Methoxy, 5'-hydroxy, Sericealactone, Sinapyl alcohol, 1-O- (3-Methyl-2-butenoyl), Sinapyl alcohol, 1-O-Angeloyl, 10- (3,4-dimethoxy-6 -methyl-2,5-benzoquinone) -9-hydroxy-4,8-dimethyl-9-hydroxy-4-decenoic acid.

  As the presumed structure of Compound No. 6, 3-O-sulfated 4,7-Megastigmadiene-3,9-diol, 4,5,7,8-Tetrahydro, 9-O-sulfated 4,7-Megastigmadiene-3, 9-diol, 4,5,7,8-Tetrahydro or 5-Megastigmene-3,9-diol, 5,6-dihydroxy, 8-Hydroxy-5-isopropyl-8-methyl-6-nonan-2- one, 9-Dodecan-1-ol, Formyl, or sulfate conjugates thereof.

  As the deduced structure of Compound No. 11, 1,3,7,14-Tetrahydroxy-16-kaurene-12,15-dione, 16,17-Dihydro, 3-Ac, 1,7,14,18,20- Pentahydroxy-16-kauren-15-one, 18-Ac, 12,20-Epoxy-11,13,20-trihydroxy-3,14-clerodadien-2-one, 3,4-Epoxide, 11-Ac, 12, 20-Epoxy-7,11,13,20-tetrahydroxy-3,14-clerodadien-2-one, 11-Ac, 15,16-Epoxy-3,4,7,12-tetrahydroxy-13 (16), 14 -clerodadien-20-al, 7-Ac, 15,16-Epoxy-6,9,13,20-tetrahydroxy-14-labden-19-oic acid, 19,6 Lactone, 20-Ac, 2,19: 4 , 18: 11,16: 15,16-Tetraepoxy-14-clerodene-6,19-diol, 14,15-Dihydro, 6-Ac, 3,4,11-Trihydroxy-6-eudesmen-8-one, 3 -(2,3-Epoxy-2-methylbutanoyl), 4-Ac, 3,4,11-Trihydroxy-6-eudesmen-8-one, 11-Hydroperoxide, 3-angeloyl, 4-Ac, 3,4,11 -Trihydroxy-6-eudesmen-8-one, 3- (2,3-Epoxy-2-methylbutanoyl), 4-Ac, 3,4-Dihydroxy-7 (11) -eudesmen-8-one, 7,11- Epoxide, 3- (2,3-epoxy-2-methylbutanoyl), 4-Ac, 3,5,7-Trihydroxy-p-menth-1-en-6-one, 6-Alcohol, 3,6-ditigloyl, 5-Ac, 4,18: 15,16-Diepoxy-13 (16), 14-clerodadiene-3,6,12,19-tetrol, 6-Ac, 7,20-Epoxy-16 -kaurene-1,6,7,11,15-pentol, 11-Ac, 7,20-Epoxy-16-kaurene-1,6,7,14,15-pentol, 1-Ac, 7,20-Epoxy -16-kaurene-1,6,7,15,19-pentol, 19-Ac, 7,20-Epoxy-16-kaurene-6,7,11,14,15-pentol, 6-Ac, 7,8 , 16,18-Tetrahydroxy-19-serrulatanoic acid; 18-Ac, 9,13-Epoxy-3,6-dihydroxy-7-oxo-15,16-labdanolide, 3-Ac, Cinncassiol A; 19-Deoxy, 1 -Ac, Dysodanthin E, Hymenoxon; 2-Tigloyloxy, 4-Et ether, Javanicin Z, Nigakilactone N; 12-Me ether, Picrasinol D.

  As the predicted structure of Compound No. 13, 3-O-sulfated 3-Hydroxy-5-megastigmene-4,9-dione, 3-O-sulfated 3-Hydroxy-5-megastigmene-7,9-dione, or 1 -Butoxy-3-phenoxy-2-propanol, 10-Hydroxy-8-dodecen-11-ynoic acid, Me ester, 11-Hydroxy-4-methyl-2,4,6-dodecatrienoic acid, 13-Oxo-9, 11-tridecadienoic acid, 3,11-Dihydroxy-5-megastigmen-9-one, 11-Aldehyde, 3,5-Dihydroxy-6,7-megastigmadien-9-one, 5,11-Epoxy-9-hydroxy-7 -megastigmen-3-one, 5,13-Epoxy-7-megastigmene-3,9-dione; 7,8-Dihydro, 5,6-Epoxy-7-megastigmene-3,9-diol, 9-Ketone, 5 , 6-Epoxy-7-megastigmene-3,9-diol, 3-Ketone, 5- (2-Hydroxy-2-methylpropylidene) -3-methoxy-2,4,4-trimethyl-2-cyclopenten-1-one , 5- (7-Hydroxy-6-methyloctyl) -2 (5H) -furanone; 7'-Ketone, 6,9-Dihydroxy-4,7-megastigmadien-3-one, 6-Hydroxy-7-megastigmene-3 , 9-dione, Guaymasol, Hyalopyrone, Jasmonic acid; Me ester, Johanilactone, Norannuic acid, Similin A, Stegobiol; 2'-Ketone, p-Mentha-1,3,5-triene-2,3,5-triol; Tri-Me ester or their sulfur And acid conjugates.

  As the presumed structure of Compound No. 14, 11-Hydroxy-9-tridecenoic acid, 7-Hydroxy-4-dodecenoic acid, Me ether, 7-Megastigmene-3,4,9-triol, 7-Megastigmene-3,6 , 9-triol, 7-Megastigmene-5,6,9-triol, 8,9-Dihydroxy-5-isopropyl-8-methyl-6-nonen-2-one, 8-Hydroxy-5-isopropyl-8-methyl -6-nonen-2-one, 6,7-Epoxide, 9,13-Dihydroxy-3-megastigmanone, Trimethyl-2- (1-methylethyl) -6,8-dioxabicyclo [3.2.1] octane-7-methanol Or these glucuronic acid conjugates, or 5,11-Epoxy-7-megastigmene-3,6,9-triol, 7-Megastigmene-2,5,6,9-tetrol, 9-Ketone, 9-Hydroxy -2,2,4-trimethyl-6-oxo-3-decenoic acid, or these O-glycosides.

  As the presumed structure of Compound No. 20, 11-Hydroxy-9-tridecenoic acid, 7-Hydroxy-4-dodecenoic acid, Me ether, 7-Megastigmene-3,4,9-triol, 7-Megastigmene-3,6 , 9-triol, 7-Megastigmene-5,6,9-triol, 8,9-Dihydroxy-5-isopropyl-8-methyl-6-nonen-2-one, 8-Hydroxy-5-isopropyl-8-methyl -6-nonen-2-one, 6,7-Epoxide, 9,13-Dihydroxy-3-megastigmanone, Trimethyl-2- (1-methylethyl) -6,8-dioxabicyclo [3.2.1] octane-7-methanol Or sulfate conjugates thereof.

  The presumed structure of Compound No. 22 includes 2-O-sulfated 2-hydroxy-dihydroactinidiolide, 3-O-sulfated 3-hydroxy-dihydroactinidiolide, 4-O-sulfated 4-hydroxy-dihydroactinidiolide or its ring-opened form. .

  The presumed structure of Compound No. 23 is 3-O-sulfated 3,9-dihydroxy-5-megastigmene, 9-O-sulfated 3,9-dihydroxy-5-megastigmene, 3-O-sulfated 3-hydroxy-5 -megastigmene-9-one, 5,6-dihydro, or 4,7-Megastigmadiene-3,9-diol, 7,8-Dihydro, 8-Hydroxy-5-isopropyl-8-methyl-6-nonen-2- one, 9-Dodecen-1-ol, Formyl or sulfate conjugates thereof.

  As the deduced structure of Compound No. 32, 4-Pregnen-19-ol-3, 20-dione is most preferable, and 4-Pregnen-6β-ol-3, 20-dione, 4-Pregnen-7α- ol-3, 20-dione, 4-Pregnen-7β-ol-3, 20-dione, 4-Pregnen-11α-ol-3, 20-dione, 4-Pregnen-11β-ol-3, 20-dione, 4-Pregnen-21-ol-3, 20-dione, 4-Pregnen-17-ol-3, 20-dione, 4-Pregnen-12α-ol-3, 20-dione, 4-Pregnen-16α-ol- 3, 20-dione, 4-Pregene-18-ol-3, 20-dione, 4-Pregne-18-ol, 3, 20-dione (20α and 20β ols) 18, 20-hemiketal, 5-Pregnen-16 , 17-epoxy-3β-ol-20-one, 5-Pregnen-3β-ol-7, 20-dione, 5-Pregnen-3β-ol-11, 20-dione, 5-Pregnen-3β-ol-16 , 20-dione, 16, (5α) -Pregnen-3α-ol-11, 20-dione, 16, (5α) -Pregnen-3β-ol-11, 20-dione, 5, 16-Androstadien-3β-ol -17β-carboxylic acid methyl ester, 1, (5α) -Androsten-17β-ol-3-one acetate, 2, (5α) -Androsten-3-ol-17-one acetate, 2, (5α) -Androsten- 11α-ol-17-one acetate, 4-Androsten-3β-ol-17-one acetate, 4-Androsten-17α-ol-3-one acetate, 4-Androsten-17β-ol-3-one acetate, 4- And rosten-3-one-17β-carboxylic acid methyl ester, 5-Androsten-3, 17-dione 3-ethylenketal, 5-Androsten-3β-ol-16-one acetate, 5-Androsten-3α-ol-17-one acetate, 5-Androsten-3β-ol-17-one acetate, 4-Estren-7α-methyl-17β-ol-3-one acetate, or 4-Estren-17β-ol-3-one propionate or its sulfate conjugate Is mentioned.

  Some of these biomarkers for stress diseases were identified as follows.

(6. Identification of biomarkers of stress-related diseases by NMR)
[6.1 Separation and purification of biomarker samples for NMR analysis and structure estimation]
After lyophilizing 10 mL of urine from the RCS-loaded medium administration group sample, it was redissolved in 300 μL of water. This redissolved sample was injected into an HPLC-MS system instrument (manufactured by Varian), and the eluate was fractionated and collected every 20 seconds. Among the collected fractions, fractions detected by mass spectrometry as m / z 289 were collected, concentrated to dryness under reduced pressure, and redissolved in 50 μL of water. Using three different types of columns, the same fraction-collection and concentration operations by HPLC-MS were sequentially performed three more times. In the fourth round, the desired fraction was concentrated to dryness under reduced pressure and then dissolved in 180 μL of deuterated methanol to prepare a sample for urine purification NMR measurement.

The HPLC conditions, MS measurement conditions, and NMR measurement conditions are as follows.
<HPLC conditions>
・ Column: (First) Unison UK-C8 (3μm particle size 4.6mm inner diameter x 250mm length) (Imtakt)
(Second) Unison UK-Phenyl (3μm particle size 6.0mm inner diameter x 250mm length) (Imtakt)
(3rd) Cadenza CD-C18 (3μm particle size 6.0mm inner diameter x 250mm length) (Imtakt)
(4th) Unison UK-Phenyl (3μm particle size 6.0mm ID x 250mm length) (Imtakt)
-Mobile phase component A: 0.1 wt% formic acid aqueous solution-Mobile phase component B: acetonitrile-Gradient conditions for elution of mobile phase component A / B Time (min) / B ratio (%): 0 min / 12% → 33 minutes / 45%
・ Flow rate: 1.0 mL / min ・ Column temperature: 35 ° C.
Sample injection volume: 50-95 μL
<MS measurement conditions>
・ Ionization method: Negative electrospray ionization method (Negative ESI)
Mass measurement range: m / z 100-500
<NMR measurement conditions>
Measuring instrument: 600MHz nuclear magnetic resonance analyzer VNMRS 600 (Varian)
・ Probe: 600MHz ¹ H { ¹³ C / ¹⁵ N} 5mmφ
PFG Triple Resonance ¹³ C Enhanced Cold Probe
・ Chemical shift standard: CD ₃ OD (3.3ppm)
Measurement method: wet ¹ -dimensional ¹ H-NMR and wet TOCSY method

The results of ¹ H-NMR measurement are shown below.
δ (ppm) 4.57 (m, 1H), 2.55 (m, 2H), 2.44 (m, 1H), 2.28 (m, 1H), 2.21 (m, 1H), 2.13 (s, 3H), 2.12 (m, 1H), 1.95 (m, 1H), 1.61 (s, 3H), 1.52 (t, 1H), 1.06 (d, 6H)

According to the NMR spectrum analysis, the structure of the compound (compound No. 16) having an m / z of 289 by mass spectrometry has a molecular formula of C ₁₃ H ₂₂ O ₅ S, a molecular weight of 290, and is represented by the following chemical formula (2). It was estimated that

[6.2 Identification of biomarkers by LC-NMR / MS]
The urine purified NMR measurement sample having the m / z of 289 was used as it was as the LC-NMR / MS measurement sample. On the other hand, a sample with the structure of chemical formula (2) obtained by synthesis separately was dissolved in deuterated methanol so as to be 2 mg / mL, and used as a sample for LC-NMR / MS measurement. Both samples were each injected into the HPLC and collected in a fraction loop using mass spectrometry m / z values. Each of the relevant fractions was measured by NMR.

<LC-NMR / MS system equipment>
・ HPLC apparatus: PRO STAR (manufactured by Varian Technologies)
NMR device: VNMRS 600 (manufactured by Varian Technologies)
・ MS device: 1200L (manufactured by Varian Technologies)
<HPLC conditions>
Column: Cadenza CD-C18 (3 μm particle size 4.6 mm inner diameter x 250 mm length) (Imtakt)
-Mobile phase component A: 0.1 vol% heavy aqueous solution of heavy formic acid-Mobile phase component B: Heavy acetonitrile-Gradient conditions for elution of mobile phase component A / B Time (min) / B ratio (%): 0 min / 25% → 15 minutes / 40%
・ Flow rate: 1.0 mL / min ・ Column temperature: 40 ° C.
Sample injection volume: 10 to 45 μL
<MS conditions>
・ Ionization method: Negative electrospray ionization method (Negative ESI)
Mass measurement range: m / z 100-500
<NMR conditions>
・ Probe: 600MHz ¹ H { ¹³ C / ¹⁵ N} 5mmφ
PFG Triple Resonance ¹³ C Enhanced Cold Probe
・ Fraction collection method: Fraction loop method ・ Chemical shift standard: TSP-d ₄ (0ppm)
Measurement method: wet ¹ -dimensional ¹ H-NMR and wet TOCSY method

The results of NMR measurement are shown below.
・ Sample for urine purification NMR measurement δ (ppm) 4.55 (m, 1H), 2.58 (m, 2H), 2.38 (m, 1H), 2.26 (m, 1H), 2.22-2.10 (m, 5H), 1.89 ( m, 1H), 1.59 (s, 3H), 1.54 (t, 1H), 1.04 (d, 6H)
Standard sample δ (ppm) 4.55 (m, 1H), 2.58 (m, 2H), 2.38 (m, 1H), 2.26 (m, 1H), 2.22-2.10 (m, 5H), 1.89 (m, 1H ), 1.59 (s, 3H), 1.54 (t, 1H), 1.04 (d, 6H)

  Since both NMR spectrum data were completely in agreement, the compound of the sample for urine purification NMR measurement was identified as the compound of the chemical formula (2).

  For other biomarker candidate compounds, (1) identification by MS / MS, or (2) using m / z value, various databases (Human metabolome database (http://hmdb.ca/labm/jsp/mlims /MSDbParent.jsp), MassBank.jp (http://www.massbank.jp/QuickSearch.html) and Metabolome.jp (http://www.metabolome.jp/mass_search/))) Estimation was performed.

(7. Examination of defecation volume and biomarker volume in restraint stress model)
Male Sprague-Dawley (SD) rats (Nippon Charles River, 7 weeks old at the time of use) were subjected to restraint stress (Journal of Pharmacolgical Sciences, 2007, Vol. 104, p. .263-273). Two hours after administration of the vehicle or test substance to rats, stress loading was initiated by placing the rats in a restraint stress gauge (W250 × L110 × H190 mm; KN468; Natsume Seisakusho). The urine of rats loaded with the restraint stress was collected over time, and the total defecation wet weight (g / h) of the rats was measured. With respect to the collected urine, the biomarker candidate compounds selected in the above examples were quantified using the UPLC / MRM method. As a result, it was found that the amount of defecation during stress loading, which is an index of stress, and the amount of biomarker of the present invention often show a positive correlation. In particular, the biomarker of Compound No. 29 showed a positive correlation of r = 0.59, suggesting an association with stress symptoms.

  Biomarkers for stress diseases according to the present invention, methods for distinguishing test drugs as therapeutic agents for stress diseases for mammals using the same, and methods for measuring the biomarkers include screening for therapeutic agents for stress diseases, preclinical development Efficacy assessment / efficacy assessment and setting of effective dose in animal models at the stage, confirmation of pharmacological response at the clinical development stage, selection of the optimal drug for each patient at the clinical site, prediction of the occurrence of drug efficacy for each patient -It is useful for medical care such as sensitivity prediction, early prediction / efficiency selection of patients showing efficacy, and confirmation of treatment effect.

Claims (8)

The following chemical formula ( 2)

In represented Ru of compounds;
及 beauty following chemical formula (6)

A sulfated conjugate of 3-methoxy-4-hydroxyphenyl glycols represented by:
A biomarker for irritable bowel syndrome , which is any of the above. The following chemical formula (2)

A compound represented by A sample of a biological fluid such as urine, blood, saliva or cerebrospinal fluid collected from a mammal suffering from irritable bowel syndrome and administered a test drug; and a mammal suffering from irritable bowel syndrome The measurement using the concentration or presence or absence of the biomarker according to claim 1 as an index for a biological sample selected from a sample obtained by adding a test drug to a cell collected from an organ or tissue or any of them. To distinguish the test drug as a remedy for irritable bowel syndrome to the mammal. Depending on the concentration of the biomarker, were selected and sensitivity and insensitivity said test drug to the mammal, or to claim 3, characterized in that estimating the effective dose of said test drug to the mammal The method described. The method according to claim 3 or 4 , wherein the index is an increase or decrease compared to the concentration of the biomarker in a sample of a living organism of the same healthy mammal. Said test drug, the method according to any one of claims 3 to 5, characterized in that a mitochondrial benzodiazepine receptor antagonists. The method according to any one of claims 3 to 6 , wherein the measurement is a mass spectrometry measurement, a liquid chromatography measurement, a gas chromatography measurement, and / or a nuclear magnetic resonance spectrum measurement. A biological body selected from urine, blood, saliva, or cerebrospinal fluid biological fluid collected from a mammal suffering from irritable bowel syndrome , and an organ or tissue extracted from the mammal, or a cell collected from any of them For a derived sample, using the concentration or presence of the biomarker according to claim 1 as an indicator, a liquid chromatographic method, a gas chromatographic method, a mass spectrometry method, a nuclear magnetic resonance spectrum measuring method, or a measuring method combining any of them To measure biomarkers of irritable bowel syndrome .

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